CRISPR-Mediated Activation of Endogenous Gene Expression in the Postnatal Heart
CRISPR-Mediated Activation of Endogenous Gene Expression in the Postnatal Heart
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DOI：
10.1161/circresaha.118.314522
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发表时间：
2019-11
期刊：
影响因子：
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通讯作者：
E. Schoger;K. Carroll;L. M. Iyer;John R. McAnally;W. Tan;Ning Liu;C. Noack;Orr Shomroni;G. Sa
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文献类型：
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作者：
E. Schoger;K. Carroll;L. M. Iyer;John R. McAnally;W. Tan;Ning Liu;C. Noack;Orr Shomroni;G. Sa
补充数字内容可在文本中找到。基本原理：通过CRISPR（成簇的规则间隔短回文重复序列）/Cas9进行的基因组编辑正在迅速发展。最近，开发了基于与转录反式激活结构域融合的核酸酶失活dead（d）Cas9的第二代CRISPR/Cas9激活系统，用于将特异性引导（g）RNA引导至任何感兴趣基因的调控区，以增强转录。迄今为止，尚未证明dCas 9在体内靶向基因组基因座中激活心肌细胞转录的应用。目的：我们的目的是开发一种心肌细胞特异性CRISPR介导的转录调节小鼠模型，并通过靶向Mef 2d和Klf 15位点（2个与心脏肥大和稳态有关的充分表征的基因）以增强转录来证明其多功能性。方法和结果：产生表达具有在Myh（肌球蛋白重链）6启动子控制下的VPR转录反式激活结构域的dCas 9的小鼠模型。这些小鼠仅在心肌细胞中无害地表达dCas 9。对于最初的概念验证，我们选择了Mef 2d和Klf 15，Mef 2d在过度表达时会导致肥大和心力衰竭，Klf 15在新生儿心脏中表达较低。最有效的gRNA首先在成纤维细胞（C3 H/10 T12）和成肌细胞（C2 C12）细胞系中鉴定。使用改进的三重gRNA表达系统（TRISPR [三重gRNA表达构建体]），同时转导多达3种不同的gRNA以鉴定转录激活的最佳条件。对于经验证的gRNA组合的体内递送，我们采用经由腺相关病毒血清型9的全身施用。关于靶向Mef 2d表达的gRNA递送，我们概括了预期的心脏肥大表型。使用靶向Klf 15的gRNA，我们可以显著增强其转录，尽管Klf 15在该时间点在生理上是沉默的。我们进一步证实了特异性和稳健的dCas 9VPR中靶效应。结论：所开发的小鼠模型允许通过使用内源性调控基因组元件来增强基因表达。在2个独立的基因组位点的概念验证表明，在控制出生后心脏的心肌细胞的转录的多功能应用。
Supplemental Digital Content is available in the text. Rationale: Genome editing by CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 is evolving rapidly. Recently, second-generation CRISPR/Cas9 activation systems based on nuclease inactive dead (d)Cas9 fused to transcriptional transactivation domains were developed for directing specific guide (g)RNAs to regulatory regions of any gene of interest, to enhance transcription. The application of dCas9 to activate cardiomyocyte transcription in targeted genomic loci in vivo has not been demonstrated so far. Objective: We aimed to develop a mouse model for cardiomyocyte-specific, CRISPR-mediated transcriptional modulation, and to demonstrate its versatility by targeting Mef2d and Klf15 loci (2 well-characterized genes implicated in cardiac hypertrophy and homeostasis) for enhanced transcription. Methods and Results: A mouse model expressing dCas9 with the VPR transcriptional transactivation domains under the control of the Myh (myosin heavy chain) 6 promoter was generated. These mice innocuously expressed dCas9 exclusively in cardiomyocytes. For initial proof-of-concept, we selected Mef2d, which when overexpressed, led to hypertrophy and heart failure, and Klf15, which is lowly expressed in the neonatal heart. The most effective gRNAs were first identified in fibroblast (C3H/10T1/2) and myoblast (C2C12) cell lines. Using an improved triple gRNA expression system (TRISPR [triple gRNA expression construct]), up to 3 different gRNAs were transduced simultaneously to identify optimal conditions for transcriptional activation. For in vivo delivery of the validated gRNA combinations, we employed systemic administration via adeno-associated virus serotype 9. On gRNA delivery targeting Mef2d expression, we recapitulated the anticipated cardiac hypertrophy phenotype. Using gRNA targeting Klf15, we could enhance its transcription significantly, although Klf15 is physiologically silenced at that time point. We further confirmed specific and robust dCas9VPR on-target effects. Conclusions: The developed mouse model permits enhancement of gene expression by using endogenous regulatory genomic elements. Proof-of-concept in 2 independent genomic loci suggests versatile applications in controlling transcription in cardiomyocytes of the postnatal heart.